Estrogen receptor alpha mutations regulate gene expression and cell growth in breast cancer through microRNAs

Abstract Estrogen receptor α (ER) mutations occur in up to 30% of metastatic ER-positive breast cancers. Recent data has shown that ER mutations impact the expression of thousands of genes not typically regulated by wildtype ER. While the majority of these altered genes can be explained by constant activity of mutant ER or genomic changes such as altered ER binding and chromatin accessibility, as much as 33% remain unexplained, indicating the potential for post-transcriptional effects. Here, we explored the role of microRNAs in mutant ER-driven gene regulation and identified several microRNAs that are dysregulated in ER mutant cells. These differentially regulated microRNAs target a significant portion of mutant-specific genes involved in key cellular processes. When the activity of microRNAs is altered using mimics or inhibitors, significant changes are observed in gene expression and cellular proliferation related to mutant ER. An in-depth evaluation of miR-301b led us to discover an important role for PRKD3 in the proliferation of ER mutant cells. Our findings show that microRNAs contribute to mutant ER gene regulation and cellular effects in breast cancer cells.


INTRODUCTION
Estrogen receptor ␣ (ER) is a steroid hormone receptor that is activ ated b y estrogens and is expressed in 70% of breast cancers. ER plays an important role in driving the progression of these tumors by regulating genes to promote growth and proliferation. Due to the availability of effecti v e anti-estro gen thera pies, including selecti v e estrogen receptor modulators and aromatase inhibitors, ERpositi v e breast cancers generally have a good prognosis with a 5-year survival rate of greater than 90% ( 1 ). Howe v er, many ER-positi v e breast cancer patients e v entually de v elop resistance to hormone therapies and experience r ecurr ent metastatic disease ( 2 , 3 ). Activating mutations in the ligand binding domain (LBD) of ER have been observed in approximately 30% of hormone therap y-r esistant ER-positi v e breast cancers (4)(5)(6). Over the last decade, several studies hav e improv ed our understanding of ER mutations and the mechanisms by which these mutations lead to breast cancer r ecurr ence (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Structural changes in ER's LBD as a result of ER LBD mutations confer an acti v e conformation and ligand-independent activity to unliganded mutant ER, which dri v es large transcriptional changes in the absence of estrogens ( 5 , 7 , 9 , 11-16 ). Se v eral studies have shown that this dramatic change in gene expression can dri v e increased cellular proliferation, invasion, and metastasis in breast cancer cells expressing mutant ER ( 4 , 5 , 9 , 13 , 14 , 16-18 ). Recently, w e show ed that over half of these gene expression changes are dri v en directly by the constituti v e acti vity of ER ( 16 ). Additionally, changes in ER's genomic binding and widespread alterations to chromatin accessibility, accompanied by binding of other transcription-regulating factors such as CTCF, FOXA1 and OCT1, can explain an additional 20-30% of transcriptional changes observed in ER mutant breast cancer cells. Howe v er, the intermediate factors driving these transcriptional changes and the causes of the remaining unexplained gene expression changes are unknown.
Since ER has been shown to activate several microR-NAs ( 19 , 20 ), we hypothesized that regulation of microR-N As (miRN As) could contribute to altered gene expression in ER mutant cells. miRNAs are short RNA molecules with an average length of 22 bp that are transcribed by RNA polymerase II. miRNAs typically bind directly to the 3 untranslated region (UTR) of mature messenger RNAs (mR-N As), w hich usuall y leads to decreased translation and increased degradation of the target mRNAs ( 21 , 22 ). Many miRNAs have been described to have either oncogenic or tumor-suppressi v e roles, and the altered expression of these miRNAs often contributes to cancer-associated cellular pr ocesses, including increased pr olifer ation, migr ation, and metastasis (23)(24)(25)(26). These effects are often produced when miRNAs alter the activity of important signaling pathways in cancer cells ( 27 ). Altered activity of these pathways by miRNAs could provide therapeutic opportunities. In addition, miRNA mimics and inhibitors have recently been considered as a potential therapeutic approach for treating some diseases ( 26 , 28-30 ). The possible use of miRNA mimics and inhibitors as cancer treatments in the future makes understanding the role of miRNAs in cancer an important subject.
Activated ER homodimerizes and binds to genomic elements in the genome, termed estrogen response elements (ERE), where it recruits cofactors that dri v e the transcription of nearby genes via RN A Pol ymerase II. Because miR-NAs are transcribed by RNA Polymerase II, they could be dir ectly r egulated by ER or by other transcription factors downstream of ER. Indeed, some miRNAs have previously been shown to be regulated b y ER activ ation ( 19 , 20 ). These ER regulated miRNAs, along with many other miR-NAs, have been shown to contribute to cancer progression in ER-positi v e breast cancers ( 27 , 31-38 ). Despite the clear role of miRNAs in contributing to cancer progression both broadly and in the specific case of ER-positi v e breast cancers, the role of miRNAs in r ecurr ent mutant ER-positi v e breast cancers has thus far remained unexplored. The potential for ER to directly or indirectly regulate miRNA transcription suggests that differential expression of miRNAs could occur in ER mutant cells and could contribute to the large transcriptional changes we and others have observed in mutant ER-positi v e breast cancer cells.
Here, we investigate the role of miRNAs in regulating gene expression in breast cancer cell lines we de v eloped previousl y, w hich express wildtype (WT) ER or heterozygously express either the Y537S or the D538G mutation, the two most common ER LBD mutations, from the endogenous ER locus ( 16 ). We identified many miRNAs that are differ entially expr essed in ER mutant cells compar ed to WT ER cells. We examined the effects of several of these miR-NAs, including miR-301b, miR-181c, miR-331, and let-7f, on gene expression and found that they directly or indirectly alter the expression of hundreds of genes previously identified as being differ entially expr essed in ER mutant cells compared to WT cells (mutant-specific genes) ( 16 ). These miRNAs also impact the ability of ER mutant cells to proliferate, a process that is significantly increased in ER mutant cells compared to their WT counterparts. In particular, we identify miR-301b as being significantly downregulated in ER mutant cells, which led to alter ed expr ession of mutant-specific genes and increased proliferation. Decreased miR-301b expression increases the RNA expression of PRKD3 , a kinase regulator of se v eral cellular signaling pa thways, including the RAS / MEK / ERK pa thway (39)(40)(41)(42). We found that inhibiting PKD3 protein leads to significantly decreased proliferation in ER mutant cells. In summary, we discovered that several miRNAs, including miR-301b, play an important role in driving ER mutant gene regulation and contribute to increased cellular proliferation in ER mutant breast cancer cells. We show that these miRNAs and their targeted pathways could provide alternati v e therapeutic strategies for treating mutant ER-positi v e breast cancer.

Cell culture
The generation of T-47D ER mutant and WT clones was described in Arnesen et al. ( 16 ). Cells were cultured in RPMI1640 Media (ThermoFisher Scientific) supplemented with 10% FBS (Sigma-Aldrich) and 1% penicillinstreptomycin (ThermoFisher Scientific). Five days prior to all e xperiments, e xcept those otherwise specified, cells were moved to growth in hormone-deprived media which was composed of phenol r ed-fr ee RPMI1640 Media (Ther-moFisher Scientific) supplemented with 10% charcoalstripped FBS (ThermoFisher Scientific) and 1% penicillinstreptomycin. Prolonged 25 day E2 treatment of WT cells was performed as described in Arnesen et al. ( 16 ).

miRN A e xpression analysis
ER mutant or WT T-47D cells were plated in 100mm dishes and grown in estrogen-depri v ed media for fiv e days prior to treatment with DMSO (0.1%) for 8 h or for ESR1 knock-down experiments, cells were treated with DMSO (0.1%) and transfected with siRNAs targeting ESR1 or a non-targeting siRNA control, as described below, for 48 h prior to RNA harvest. Cells were washed with phospha te-buf fered saline (PBS; ThermoFisher Scientific) and treated with RLT plus (Qiagen) lysis buffer with 1% beta-mercaptoethanol (BME; Sigma-Aldrich) for cell lysis. Cells were passed through a 21-gauge needle (Sigma-Aldrich) to facilitate genomic DN A l ysis. For PDX lines, tissue samples were used from se v en PDX lines (WT:  HCI-003 and HCI-011; L536P: HCI-005, HCI-006, HCI-007; Y537S: HCI-013, HCI-013EI). PDX samples were surgically removed from mice and flash frozen. PDX samples were not trea ted. Approxima tely 20 mg of tissue per sample was homogenized in RLT plus lysis buffer with 1% betamercaptoethanol using gentleMACS M-tubes and a gen-tleMACS Octo Dissociator (Miltenyi Biotec). RNA extraction for both T-47D and PDX samples was performed using a Quick-RN A RN A Minipr ep kit (Zymo Resear ch), and RNA was measured using a Qubit fluorometer. For each sample, 200 ng of total RNA was submitted for miRNA expression analysis using NanoString's nCounter Human v3 miRNA expression assay. NanoString miRNA expression assay results in the form of raw counts were analyzed using NanoString's nSolver software. Raw counts were normalized using two approaches. The first approach used the geometric mean for each sample of the top 100 highest expressed miRNAs across all samples to determine a normalization factor for each sample. The second approach used the geometric mean for each sample of the fiv e spike-in miRNAs profiled for the assay to generate the nor malization factors. Nor malized counts were then logscaled and used to perform t tests to identify significantly differ entially expr essed miRNAs. Because this was a hypothesis generating step, we considered differentially expressed miRNAs from both normalization approaches in our study. A paired t test was used for calling differentially expressed miRNAs from the ESR1 knock-down experiment due to clonal variability between clones of the same genotype. The DIANA TarBase database ( 43 ) was used to compare putati v e target genes of differentially expressed miRNAs with mutant-specific genes described in Arnesen et al ( 16 ). Significant enrichments were defined using a hypergeometric test as implemented in R using phyper.

Cell transfections
After cells were grown in hormone-depri v ed media for fiv e days, cells were transfected with a miRNA mimic, miRN A inhibitor, or an siRN A. mirVana miRN A mimics and inhibitors were used for 15 miRNAs (ThermoFisher Scientific). Mimics and inhibitors were transfected at a 10nM final concentration using the RNAiMAX transfection reagent (ThermoFisher Scientific) per the manufacturer's instructions. siRNAs against PRKD3 (Integrated DN A Technolo gies' TriFECTa RN Ai kit) or against ESR1 (Horizon siGENOME SMARTPool siRNA reagents, M-003401-04-0005) were used to knock-down PRKD3 or ESR1 transcript le v els, respecti v ely (sup. tab le S4). To test the efficacy of these siRNAs, WT cells were transfected with 0.1nM, 1nM, or 10nM concentrations of three anti-PRKD3 or the pooled anti-ESR1 siRNAs using the RNAiMAX transfection reagent. The two siRNAs achieving the best knock-down of PRKD3 or the pooled anti-ESR1 siRNAs were used at a 10nM concentration in subsequent siRNA transfection experiments.

Pr oliferation assa ys
Cells were plated in 100mm dishes and grown in hormonedepri v ed media for four days prior to plating in 96-well pla tes a t approxima tely 12500 cells per well and allowed to adhere for another day in hormone-depri v ed media. Twenty-four hours after 96-well plating, cells wer e tr eated with 1% DMSO and a miRN A mimic / inhibitor, siRN A, or drug, depending on the experiment. Cells were then placed on the IncuCyte Zoom Li v e Cell Imaging Platform (Sartorius) and proliferation was monitored for 48 h with 10 × ma gnification ima ges obtained at 2-h intervals. Confluence was measured for three or four replicates, depending on the experiment, and percent confluence values were normalized to the initial confluence and then log 2 transformed. Linear r egr ession analysis using a general linear Wald test, as implemented by glm() in R, was used to compare the slopes of transformed confluence values over time and to determine significant differences in slopes.

RNA-seq
T-47D cells were grown in 100 mm plates in hormonedepri v ed media for four days then plated into 12-well plates a t approxima tely 50% confluence and allowed to adhere for 24 h. Cells were then transfected with a miRNA mimic or inhibitor for a gi v en miRNA or with a mimic or inhibitor negati v e control at a 10 nM final concentration. Twenty-four hours post-transfection, cells were collected, washed with PBS, then treated with RLT Plus lysis buffer supplemented with 1% BME. RNA was extracted using a Quick-RNA RNA Miniprep kit (Zymo Research), and RNA was measured using a Qubit fluorometer. Poly-A selected RNA-seq libraries were generated using a KAPA Stranded mRNA-Seq kit (KAPA Biosystems) and 500 ng RNA per sample as starting material. Libraries were sequenced using the Illumina NovaSeq 6000 platform. The Hisat2 spliced aligner was used to align the resulting fastq files to the hg19 human genome build ( 44 ). SAM files were then converted to BAM files using SAMtools ( 45 ). Genes included in the Uni v ersity of California Santa Cruz's (UCSC) Known Genes table were assigned counts using the Subread package's featureCounts program using BAM files as inputs ( 46 ). Counts were then normalized and analyzed for differential expression using the DESeq2 package in R ( 47 ). Two clones for each ER genotype (Y537S, or D538G) were used for RNA-seq and clones of the same genotype were used as biological duplicates in our differ ential expr ession analysis. Significantly up-or down-regulated genes (adjusted Pvalue < 0.05) were identified by comparing results from miRNA mimic or inhibitor treatment to the appropriate negati v e control treatment in clones of the same ER genotype. Genes that were identified as differentially expressed wer e compar ed to mutant-specific genes as defined pr eviousl y ( 16 ). miRN A-altered m utant-specific genes were compared with putati v e miRNA target genes for the corresponding miRNA using the miRDB microRNA Target Prediction Database ( 48 ). Significant enrichments were determined using a hypergeometric test as implemented in R using phyper with a P -value cutoff of < 0.05. Gene ontology and pathway analysis was performed by submitting genes on interest to Ma'ayan lab's Enrichr online tool ( 49 ). Adjusted P -values were used to identify significantly enriched ontologies and pathways. Venn diagrams were made using the eulerr package.

Quantitative PCR
WT T-47D cells were grown in regular media. Cells were lysed using RLT plus (Qiagen) plus 1% betamercaptoethanol (Sigma-Aldrich) and total RNA was extracted using a Quick RNA Miniprep kit (Zymo Research). qPCR was performed using a Power SYBR Green RNAto-CT 1-Step Kit (ThermoFisher Scientific) and 50 ng of RNA per sample as input. A CFX Connect Real-Time light cycler (Bio-Rad) was used for thermocycling and light detection. A C t method was used to calculate expression le v els with TBP used as the control. Duplicates were measured for each sample. Primer sequences for PRKD3 and TBP primers are listed in Supplemental Table S4. Significant changes in relati v e e xpression were determined using a one-tailed Student's t test using t.test in R, with a P -value cutoff of < 0.05.

Drug treatments
Cells were grown for a single clone for each ER genotype (WT, Y537S and D538G) in 100mm dishes in hormonedepri v ed media for 4 days and then plated in 96-well plates a t approxima tely 12 500 cells per well and allowed to adhere in hormone-depri v ed media for 24 h. Cells were then treated with varying doses (0.01, 0.1, 0.5, 1, 5, 10 or 100 uM) of the PKD inhibitor CRT0066101 (Selleck Chemicals, S8366) or with a vehicle control (H 2 O) and analyzed for proliferation. Experiments were performed in triplicate. Prolifer ation r ates were calculated as described above and IC 50 values were calculated using Prism 9 software.

Statistical analysis and packages
All statistical analyses, except those for gene ontology and pathwa ys analysis, were perf ormed using R version 3.5.2 or 3.5.3.

ER mutations alter the expression of miRNAs
Previously, we identified thousands of genes that are differ entially expr essed in D538G or Y537S ER mutant cells compared to WT ER cells (mutant-specific genes) regardless of the presence or absence of 17 ␤-estradiol (E2). Posttranscriptional mechanisms, such as differential expression of miRNAs in ER mutant cells, may contribute to mutantspecific gene expression. To determine whether there is differ ential expr ession of miRNAs in ER mutant cells, we utilized T-47D breast cancer cells, de v eloped pre viousl y, w hich heterozygously express either the D538G or Y537S mutant ESR1 allele from the endogenous ESR1 locus ( 16 ). ER mutant and WT cells were grown for 5 days in estrogendepri v ed media followed by RNA extraction. Total RNA was profiled for miRNAs using the Nanostring Human v3 miRN A Assay, w hich examines the expression of nearly 800 miRNAs. We found that 84 miRNAs are differentially expressed in the D538G, Y537S, or both mutant cell lines compared to WT cells ( Figure 1 and Supplementary  Table S1).
To identify which differentially expressed miRNAs likely contribute to mutant-specific gene expression, we utilized the DIANA-TarBase v8, an open-access r efer ence database of experimentally supported miRNA targets ( 43 ). We compared the target genes for each differentially expressed miRNA with our previously identified mutantspecific genes. Because miRNAs effecti v ely down-regulate the expression of their target genes by promoting their degradation, we compared the target genes of miRNAs that exhibited increased expression in ER mutant cells to mutant-specific down-regulated genes and the target genes of miRNAs that wer e down-r egulated in ER mutant cells to mutant-specific up-regulated genes. Using this approach, we identified 15 differentially expressed miRNAs whose target genes are significantly enriched for mutant-specific genes (  these 15 miRNAs, 2 (miR-301b and miR-489) were differentially expressed in both the Y537S and the D538G mutant cells, while the remaining 13 miRNAs were differentially expressed in Y537S cells only (8 miRNAs) or in the D538G cells only (5 miRNAs). We found that genes that ar e both differ entially expr essed in ER mutant cells and are target genes of these 15 differ entially expr essed miR-NAs are enriched for a number of cellular processes such as increased proliferation and cell migration (Supplementary Figure 1A-D). These processes were pre viously observ ed to be significantly altered in ER mutant cells. In particular, increased cell proliferation was a strong characteristic of both Y537S and D538G ER mutant cells ( 16 ).
The differential expression of these 15 miRNAs, as well as the remaining 69 differentially expressed miRNAs, occurs as a result of mutant ER expression and could be directly regulated by ER. To determine the role of ER in regulating these miRNAs we took two approaches. We first treated WT ER cells with E2 for 25 days, recapitulating long-term ER activity, and performed miRNA expression analysis. We found that only 12 of the 84 mutant ER regulated miRNAs wer e similarly differ entially expr essed upon long-term ER activation (Supplementary Figure S2A). Of the 15 miRNAs identified above, 5 were found to be regulated by long-term E2 treatment in WT cells. We next tested the role of ER in regulating miRNAs by knocking down ER expression using siRN As. siRN A tr eatment r educed ER protein le v els in mutant cells by over 50% (Supplementary Figure S2B). We performed miRNA expression analysis on mutant ER cells after ER knock-down and found that only 7 out of the 84 m utant-regulated miRN As were significantl y differentiall y regulated in the expected direction, with the low number of significant miRN As likel y the result of high clone variation (Supplementary Figure S2C). Howe v er, nearly half of the 84 miRNAs that were differentially expressed in mutant ER cells exhibited expression changes in the expected direction upon ER knock-down, suggesting that mutant ER is likely playing a direct role in the d ysregula tion of se v eral miRNAs.
Using WT and mutant ER ChIP-seq data ( 16 ), we further investigated the potential contribution of mutant ER in regulating the expression of miRNAs. Of the 15 miR-NAs identified above as likely contributors to mutant ER gene expression, 12 had at least one ER-binding site (ERBS) within 10 kb and 13 had at least one ERBS within 100 kb of their respecti v e pri-miRNA TSS ( 50 ). Three of these miR-NAs had two or more ERBS within 10 kb while 12 had two or more ERBS within 100kb of their pri-miRNA TSS (Supplementary Figure S2D). Additionally, four of these miR-NAs had ERBS within 100 kb that exhibited significantly incr eased or decr eased ER binding in ER mutant cells compared to WT cells. Of the 12 miRNAs tha t had a t least one ERBS within 10 kb of the pri-miRNA TSS, 7 were found to exhibit moder ate, y et non-significant changes in expression upon ER knock-down in the expected direction. We also utilized previously obtained ATAC-seq data and found tha t dif ferential chroma tin accessibility near dif ferentially expressed miRNAs may also factor into the altered expression of 6 out of 15 miRNAs (Supplementary Figure S2D). These findings suggest that the differ ential expr ession of many of these miRNAs is at least in part directly controlled by mutant ER and possibly downstream factors. miRNAs located in the introns of protein-coding genes can be transcribed from their host gene promoter and expressed as a byproduct of the host gene transcription ( 51 ).
To determine the contribution of host gene differential expression to miRNA regulation, we compared the expression patterns of differ entially expr essed intronic miRNAs to the expression of their host genes as observed previously ( 16 ). We found that only 4 of 30 intronic miRNAs e xhibited e xpression pa tterns tha t coordina ted with the expression of their host genes in ER mutant cells compared to WT cells (miR-489 / CALCR, miR-1180 / B9D1, miR-140 / WWP2, and miR-15b / SMC4). These results suggest that the altered expression of most intronic miRNAs is independent of host gene regulation.

Differ entially r egulated miRNAs in ER mutant cells significantly impact cell proliferation
To more directly determine the effects of differential miRNA expression on cell proliferation in ER mutant cells, we used miRNA mimics to increase a miRNA's effect or miRNA inhibitors to decrease a miRNA's effect in both ER WT and ER mutant cells. WT or ER mutant cells were transfected with a single miRNA mimic or inhibitor for each of the 15 differentially expressed miRNAs that exhibited enrichment for mutant-specific genes in their respecti v e target gene sets. Immediately following inhibitor or mimic introduction, cells were monitored for proliferation for 48 h using the IncuCyte Zoom platform for li v e-cell imaging. Treatment with a miRNA mimic or inhibitor led to significant changes in proliferation rates in mutant and / or WT ER cells for se v eral of the 15 miRNAs examined, with miR-301b-3p, miR-181c-5p and miR-331-3p producing particularly notable effects ( In Y537S and D538G ER mutant cells, where miR-301b is significantly down-r egulated, tr eatment with a mimic for miR-301b in Y537S and D538G ER mutant cells had a striking effect, completely abr ogating pr oliferation in both ER mutant models (Figure 2 A, Supplementary Figur e S3A). Tr eatment with a miR-301b inhibitor in mutant cells increased proliferation in Y537S ER mutant cells but had no measurable effect on D538G mutant ER cells. Conversely, treatment of WT ER cells with a miR-301b inhibitor, recapitulating the loss of miR-301b in ER mutant cells, or with a miR-301b mimic had negligible effects on cell proliferation. miR-301b has been shown in other contexts to drive increased proliferation ( 52 , 53 ). However, these results suggest that the miR-301b-mediated effects on proliferation are dependent on the cells' ER mutation status and that loss of miR-301b contributes to increased prolifera tion ra tes specificall y in ER m utant cells.
Treatment with mimics or inhibitors for the other two miRNAs of interest led to milder effects on proliferation. Introduction of a mimic for miR-181c, which was downregulated in D538G mutant cells, significantly decreased proliferation in both D538G and WT ER cells ( Figure 2B; Supplementary Figure S3A). Howe v er, further decreasing the activity of miR-181c in D538G mutant cells by treating with a miR-181c inhibitor had no significant effect on proliferation (Supplementary Figure S3A). In WT ER cells, decreasing miR-181c activity via miR-181c inhibitor treatment, recapitulating the loss of miR-181c seen in D538G cells, led to a slight yet significant increase in proliferation (Figur e 2 B). Incr easing the activity of miR-331, which was down-regulated in the Y537S mutant, resulted in a significantly decreased prolifer ation r ate in both Y537S mutant and WT ER cells, while treatment of Y537S mutant or WT cells with a miR-331 inhibitor increased proliferation in both cell lines, although the effect in WT cells was moderate ( Figure 2C; Supplementary Figure S3A). The mimic or inhibitor treatments for the remaining 11 miRN As, w hich were organized into fiv e groups based on their expression patterns in WT and ER mutant cells, resulted overall in weaker proliferati v e effects (Supplementary Figure S3B-C). Together, these data show that multiple miRNAs differentially expressed in ER mutant cells contribute to the regulation of cell proliferation, a phenotype that is increased in ER mutant cells. In the ER mutant setting, these miRNAs changes dri v e increased proliferation with some miRNAs having a stronger effect on proliferation than others.

Differ entially expr essed miRNAs contribute to mutantspecific gene regulation
We investigated the effects of miR-301b, miR-181c, and miR-331, which impacted proliferation, as well as let-7f, which was exceptionally up-regulated in Y537S mutant cells, on gene expression in ER mutant cells by performing RNA-seq on total RNA collected from ER mutant or WT cells after treatment with a single miRNA mimic or inhibitor. miRNAs that were down-regulated were evaluated with a miRNA mimic (miR-301b-3p , miR-181c-5p , and miR-331-3p), while let-7f was evaluated with a miRNA inhibitor. In this way, the activity of the each miRNA of interest was effecti v ely re v erted towar ds WT le v els. RNA-seq was performed 24 h post-transfection. We found that alter ed expr ession of these miRNAs by mimic or inhibitor tr eatment r esulted in the differential expression of tens to hundreds of genes (miRNA-altered genes) depending on the miRNA (Figure 3 A and Supplementary Table S2). To valida te tha t the observed gene expr ession changes ar e a r esult of mimic or inhibitor   treatment, we asked whether miRNA-altered genes exhibited enrichment for target genes of the corresponding miRNA. Mimic treatment should lead to enrichment of miRNA tar gets in do wn-regulated genes, while inhibitor treatment should exhibit enrichment of miRNA targets in up-regulated genes. As expected, we observed an enrichment for predicted miRNA target genes in down-regulated genes altered upon miR-301b, miR-181c, or miR-331 mimic treatments and in up-regulated genes after let-7f inhibitor tr eatment (Supplementary Figur e S4A). We found that inhibition of let-7f resulted in very few gene expression changes ( n = 16), potentially due to the high expression of this miRNA in Y537S ER cells, although the affected genes were nearly all previously identified as let-7f target genes. The enrichment for miR-331 targets in down-regulated genes after miR-331 mimic treatment was relati v ely weak. Howe v er, more miR-331 target genes were observed in the group of down-regulated genes than in the group of up-regulated genes suggesting that the differential gene expression observed after mimic treatment may still be dri v en by altered miR-331 activity and that the target genes of miR-331 may not be well defined or could be highly cell type specific.

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To determine whether our miRNAs of interest contribute to the mutant-specific gene expression patterns we observed previously ( 16 ), we asked whether miRNA-altered genes were significantly enriched for mutant-specific genes. Because mimic or inhibitor treatment re v ersed the activity of these miRNAs relati v e to their e xpression in ER mutant cells compared to WT cells, we expected to find significant overlaps between mimic-or inhibitor-regulated genes and mutant-specific genes regulated in the opposite direction. For example, we would expect genes down-regulated by miR-301b mimic treatment to be up-regulated in ER mutant compared to WT cells. As expected, we found tha t genes tha t wer e up-r egulated upon mimic or inhibitor tr eatment wer e significantly over-r epr esented in the corr esponding mutant-specific down-regulated gene sets (Figure 3 B). Conversely, genes that were down-regulated upon mimic or inhibitor treatment were significantly enriched for mutant-specific up-regulated genes. Comparison of mutantspecific genes regulated in the same direction as miRNAaltered genes (i.e. mutant-specific up-regulated vs miRNA mimic / inhibitor up-regulated) resulted in non-significant or very low enrichment between gene sets (Figure 3 B). These results show that miRNAs regulate the expression of many mutant-specific genes in the expected direction and suggest that they impact gene regulation and proliferation in ER mutant cells.
The majority of mutant-specific gene expression changes can be explained by mutant ER's constitutive activity, altered ER genomic binding, or altered chromatin accessibility ( 16 ). Howe v er, the mechanism behind the differential expression of the remaining mutant-specific genes was unexplained. Our findings showed that miRNAaltered mutant-specific genes are represented in each of these categories at a distribution similar to that observed for all mutant-specific genes (Supplementary Figure  S4B). miRNAs contribute considerably to these previouslyunexplained mutant-specific gene expression effects. We found that 4 miRNAs (miR-301b, miR-181c, miR-331, and let-7f) together could explain between 18% and 34% of mutant-specific genes that were unexplained from our previous findings (D538G: 18.7%; Y537S: 33.2%; Supplementary Table S3), indicating that miRNAs could be playing a substantial role in shaping ER mutant-dri v en gene e xpression patterns.
We found that one of the mutant-specific genes regulated by miRNAs is ESR1 , the gene encoding ER. miR-301b and miR-181c were both found to regulate ER, which has been shown to have complementary sequence in its 3`UTR to the seed sequences for both of these miRNAs ( Figure 3C; Supplementary Figure S4C) ( 48 , 54 ). ER is significantly upregulated in ER mutant cells, where ER is constituti v ely acti v e, compared to WT cells (Figure 3 C). This is counter to some reports that show that ER, in the mammary gland, regulates its own expression through a negati v e feedback mechanism in which ER activity leads to decreased transcript le v els of ESR1 (55)(56)(57). This suggests that ER mutant cells are able to evade the nega tive regula tion typically exerted by active ER and potentially take advantage of the resulting sustained or e v en heightened le v els of ER to promote increased cell growth and proliferation. The observation that multiple miRNAs targeting ESR1 are significantly down-regulated in ER mutant cells indicates an ER mutantdri v en down-regulation of miRNAs as a key mechanism for evading normal negative feedback regulation by active ER on ESR1 expression. Consistent with our observations, we previously found that metastatic breast cancer patient samples harboring D538G or Y537S mutant ER exhibit significantly increased le v els of ER compared to WT ER breast cancer metastases ( 17 ).
To examine the overall effects of miRNAs in regulating ER mutant phenotypes, we performed gene ontology analysis on miRNA-altered mutant-specific genes. This analysis re v ealed that these genes play roles in se v eral important biological processes that ar e alter ed in mutant cells. miR-301b and miR-331 altered mutant-specific up-regulated genes were enriched for processes related to cell cycle progression (Figure 3 D). Many of these genes, such as CDK1 and KIF14 , play important roles in mediating cell cycle progression, indicating again the role of these miRNAs in driving increased proliferation (Figure 3 C). miRNAalter ed mutant-specific up-r egulated genes r esulting from miR-181c mimic treatment in D538G cells were also involved in cell cycle regulation, although these genes were more enriched in DNA damage repair (Figure 3 D). Additional gene ontology terms associated with miRNA-altered mutant-specific genes include cell-matrix adhesion and negati v e regulation of cell motility (Figure 3 D). These genes wer e down-r egulated in ER mutant cells and up-regulated in cells treated with either a miR-301b, miR-181c, or miR-331 mimic, suggesting that these miRNAs restrict cell motility. The decreased expression of these miRNAs in ER mutant cell lines may contribute to increased cell motility and migration observed in ER mutant cells ( 9 , 14 , 17 , 58 , 59 ).
To understand the direct effects of miRNAs in driving these broad phenotypic effects, we asked which miRNAaltered mutant-specific genes are predicted direct miRNA targets based on the miRDB database for miRNA target genes, which uses an algorithm trained on large RNAseq and CLIP-liga tion da ta sets to predict miRNA targets ( 48 ). We found that miRN A-altered m utant specific genes were enriched for miRNA target genes in the expected direction. Of the genes overlapping mutant-specific upregulated genes, 23% of miR-301b mimic ( P -value = 1.7e-26 (Y537S) and 1.2e-64 (D538G); hypergeometric test) and 48% of miR-181c mimic ( P -value = 2.1e-108; hypergeometric test) down-regulated genes were direct miR-301b or miR-181c target genes (Figure 4 A, Supplementary Figure  S5A). All of the let-7f inhibitor up-regulated genes shared with mutant-specific down-regulated genes were let-7f direct targets (Supplementary Figure S5A). Howe v er, only 1.5% ( P -value = 0.45; hypergeometric test) of miR-331 mimic down-regulated mutant-specific up-regulated genes wer e dir ect miR-331 target genes, similar to the lack of miR-331 target gene enrichment described above (Supplementary Figure S5A). We found that direct target genes of these miRNAs were enriched for se v eral key cell signaling pathways including PI3K signaling, nuclear receptor signaling, and EGF receptor signaling, specifically through the MAPK signaling cascade ( Figure 4B; Supplementary Figure S5B).

miR-301b target PRKD3 contributes to increased proliferation in ER mutant cells
Analysis of miR-301b direct target genes that were also mutant-specific genes found that many of these shared genes are key components of major cell signaling pathways including MAPK signaling, PI3K signaling, and ER signaling. Of note, miR-301b targets ERK2 , a central component of MAPK signaling which is known to dri v e cell proliferation, dif ferentia tion, and survival ( Figure 5 A), re vie wed in ( 60 ). miR-301b also targets PIK3CB (P110-␤), the catalytic subunit of PI3K ␤, which has been shown to enhance cell pr oliferation and pr omote tumorigenesis in breast cancer ( Figure 5 A), re vie wed in ( 61 ). The regulation of these pathway components by differentially expressed miRNAs in ER mutant cells indicate that altered miRNA expression in ER m utant cells directl y alters important cellular pathways with roles in regulating cell growth and proliferation. miR-301b regulates the RNA le v els of PRKD3 (the gene encoding protein kinase D3 --PKD3), which regulates acti vity of se v eral signaling pa thways by phosphoryla ting key pathway components leading to increased proliferation and cell migration (39)(40)(41)(42). PRKD3 gene expression is significantly up-regulated in both Y537S and D538G ER mutant cells but is significantly down-regulated upon treatment with a miR-301b mimic (Figure 5 A). PRKD3 contains a recognition sequence for miR-301b in its 3 UTR indicating that it is likely a miR-301b target gene (Figure 5 B) ( 54 ). Based on these observations, we assessed whether PRKD3 is important for proliferation of ER mutant cells in a similar manner to miR-301b mimic treatment. We knocked down PRKD3 expression, and found tha t trea tment with either of two anti-PRKD3 siRNAs significantly reduced PRKD3 expression and cell proliferation in ER mutant and WT cells ( Figure 5C; Supplementary Figure S6A-B). Because knock-down of PRKD3 via siRNA reduced prolifera tion, we investiga ted w hether pharmacolo gical inhibition of PKD3 with CRT0066101 could similarly reduce cell proliferation. CRT0066101 is a pan-PKD inhibitor that has been shown to reduce proliferation in a number of tumor types, including ER-negati v e br east cancer, pancr eatic cancer, and colon cancer (62)(63)(64)(65). We treated ER mutant and WT cells with CRT0066101 at a range of doses and found that CRT0066101 treatment reduced cell proliferation in all cell lines in a dose-dependent fashion ( Figure 5D; Supplementary Figure S6C). Significant reduction in proliferation rates was seen at concentrations as low as 0.1 uM, with IC 50 values between 0.5 and 1.1 uM depending on ER muta tion sta tus, similar to IC 50 values observed in other successful treatment settings ( 62 , 64 ). Significant loss of proliferation upon knock-down or inhibition of PRKD3 points to PRKD3 as a promising therapeutic target in ER-positi v e breast cancers, including those containing ER mutations.
Unexpectedly, we found that while miR-301b perturbations impacted RNA le v els of PRKD3 , we only observed minimal effects on PKD3 protein le v els with miR-301b mimics or inhibitors (Figure 5 E). It is unclear why the effects on PRKD3 RNA le v els do not correspond to PKD3 protein le v els, although this finding suggests that PKD3 protein may not play an important role in the miR-301b gro wth phenotype. Ho we v er, the r esults ar e still consistent with PKD3 r epr esenting an important factor that promotes ER mutant and WT breast cancer cell growth.

Differ ential expr ession of miRNAs in mutant ER cell lines is recapitulated in patient-derived xenograft models
To determine if the differential expression of miRNAs seen in the cell line models is observed in additional models, we used patient-deri v ed x enograft (PDX) br east cancer models ( 66 ), harboring either WT ER or naturally-occurring heterozygous Y537S or L536P ER mutations. PDX models included two WT ER lines, a series of three L536P ER mutant lines from the same patient, and two Y537S ER mutant lines from the same patient. Results of miRNA profiling re v ealed that over 400 miRNAs were significantly differentially expressed between the Y537S and WT PDX lines, w hile nearl y 60 miRNAs were significantly differentially expressed in the L536P PDX lines vs WT ( Figure 6A; Supplementary Table S1). When compared to miRNAs that are differentially expressed in the T-47D ER mutant cell lines, only one upregulated miRNA was shared between the Y537S PDX lines and the T-47D Y537S cell lines. Howe v er, half of the downregulated miRNAs identified in T-47D Y537S mutant ER cell lines were also significantly down-regulated in Y537S PDX lines (Figure 6 B). Of the 15 miRNAs that were differ entially expr essed in T-47D cells and exhibited significant overlap of target genes with mutant-specific genes, 10 wer e differ entially expr essed in the T-47D Y537S mutant clones. Of these, 5 exhibited significant up-or downregulation in the Y537S PDX lines in the same direction as that observed in the T-47D Y537S mutant clones, including miR-301b and miR-331, while an additional 3, including miR-181c and let-7f, were trending in the same direction but were below the significance threshold ( Figure  6 C-D). miRNA let-7f-5p was up-regulated with a nearly significant P -value, while miR-181c was not differentially regulated in T-47D Y537S mutant cell lines and ther efor e was not expected to be differ entially r egulated in Y537S PDX lines. Of the 5 miRNAs that wer e differ entially r egulated in the T-47D D538G m utant cell lines onl y, none were  differ entially r egulated in the PDX Y537S lines, indicating consistent ER allele-specific effects. These miRNA expression results in WT and mutant ER PDX lines show that miRNAs are significantly altered in patient-deri v ed models harboring mutant ER and validate many of our findings in T-47D cells.

DISCUSSION
The frequent occurrence of ER mutations in hormone therap y-r esistant ER-positi v e metastatic breast cancer has led to intense r esear ch ef forts to elucida te the molecular and phenotypic effects of ER mutations and to identify vulnerabilities in ER mutant cancers that could be exploited to de v elop effecti v e ther apies for hormone ther ap y-r esistant tumors (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). In a previous study, we used isogenic T-47D models expressing the Y537S or D538G ER mutations from the endogenous ER locus to show that ER mutations regulated the differential expression of thousands of genes and that these transcriptional changes were accompanied by phenotypic changes, including increased migration, invasion, and proliferation ( 16 , 17 ). Although our understanding of ER mutations and their effects has improved significantly, the contribution of miRNAs to ER mutant gene regulation and downstream effects was unknown. In humans, over 2000 miRNAs regulate the expression of roughly 60% of protein coding genes ( 67 , 68 ), indicating a potential for miRNAs to aid mutant ER in altering gene expression. In this study, we aimed to determine the role of miRNAs in regulating transcriptional and phenotypic changes in mutant ER breast cancer. Using the isogenic cell line models introduced previously ( 16 ), we found that 84 miRNA (over 10% of those profiled) wer e differ entially expr essed in the Y537S , D538G , or both mutant cell lines. Mutant-specific genes identified in our previous work were over-r epr esented in the target genes of many of these miRNAs. In se v eral cases, namely miR-301b, miR-181c, miR-331, and let-7f, these miRNA targets were enriched for genes with important roles in driving cell gr owth, pr olifera tion, or motility, indica ting a possible role for these miRNAs in driving cancer-associated phenotypes that we observed previously in ER mutant cells. When we directly tested the effect of these miRNA on ER mutant-specific gene expression by altering miRNA activity through mimic or inhibitor treatment followed by RNAseq, we found that these miRNAs did indeed alter the expression of many mutant-specific genes. Furthermore, these miRN A-altered m utant-specific genes were enriched for cell cycle-related genes and therefore altered activity of these miRNAs in ER mutant cells can partially explain the observed increased proliferation.
Alter ed expr ession of miRN As in ER m utant cells can lead to broad gene expression changes both through direct miRNA targeting or by downstream effects in which miRN As directl y alter transcriptional regulators which in turn alter the expression of their target genes. We found that for three of the four miRNAs we investigated in depth (miR-301b, miR-181c, and let-7f), mutant-specific genes that were up-or down-regulated upon miRNA inhibitor or mimic tr eatment, r especti v el y, were highl y enriched for pr edicted dir ect target genes of the respecti v e miRNA. For miR-301b and miR-181c, many direct target genes were components or regulators of important cell signaling pathways. Alter ed expr ession of these pathways could lead to additional gene expression changes, potentially explaining the remaining 50-75% of miRNA-altered mutant-specific genes. Only 2 of the 17 genes up-regulated by let-7f inhibitor tr eatment wer e also mutant-specific down-r egulated genes, Relati v e growth is shown for WT, Y537S and D538G ER clones after treatment with CRT0066101 (pan-PKD inhibitor). ( E ) Immunoblot analysis shows PKD3 expression after miR-301b inhibitor treatment in ER WT cells and miR-301b mimic in ER mutant cells. Bar graph shows quantification of PKD3 signal for two clones for each ER genotype. Error bars indicate average signal ± SEM. but both of these genes (TUSC2 and PLAGL2) were predicted let-7f direct target genes. These findings indicate that miRN A-mediated m utant-specific gene expression changes occur as a result of both direct miRNA-mediated mRNA degradation and through secondary effects dri v en by alter ed expr ession or activity of genes that ar e dir ect miRNA targets.
One major question regarding differentially expressed miRN As is w hat dri v es their altered expression. Altered miRNA expression could be driven directly by ER, by other transcription factors downstream of ER signaling, or by other transcription factors that have improved or decreased access to genomic regions due to mutant ER dri v en changes in chromatin accessibility ( 16 ). miRNA expression analysis  following long-term E2 treatment in WT ER cell lines indica ted tha t less than 15% of m utant ER regulated miRN As ar e r egulated b y long-term activ ation of WT ER. Not only does mutant ER likely regulate the expression of these miRN As, but conversel y, at least two differentially expressed miRNAs (miR-301b and miR-181c) regulate the expression of ESR1 , the gene encoding ER. miR-301b and miR-181c ar e down-r egulated in ER mutant cells which corr elates with incr eased expr ession of ER. We found that reversing the low expression of these miRNAs to higher levels, such as those observed in WT cells, through miRNA mimic tr eatment, r esulted in significantly decreased ESR1 expression. In addition, ESR1 contains recognition sequences for both miR-301b and miR-181c in its 3 UTR, indicating that ESR1 is a direct target of these miRNAs. In the normal mammary gland, ER activity results in a negative feedback loop that r educes ER expr ession ( 56 ). In T-47D cells, initial ER activity increases ER expression, but with long-term ER acti vity, ER e xpression plateaus and drops back towards starting le v els. Mutant ER-dri v en down-regulation of miR-301b and miR-181c r epr esents a mechanism by which mutant ER evades this normal negative feedback on ER expression and instead achie v es higher ER le v els.
In addition to understanding the role of miRNAs in regulating mutant-specific gene expression, we also aimed to understand how miRNAs contribute to cancer-associated phenotypes in ER mutant cells. One major phenotype observed in ER mutant cells is their increased proliferative capacity ( 16 ) and miRNAs could contribute to the positi v e effect mutant ER has on prolifera tion. W hen we tested the effect of 15 differentially expressed miRNAs on proliferation by individually modulating their activity with miRNA mimics or inhibitors, we found that se v eral miR-N As significantl y altered proliferation. In particular, miR-301b, miR-181c, and miR-331 significantly altered ER mutant cell growth. This effect is explained by the finding that the mutant-specific genes that are altered by these miRNAs include many cell cycle-related genes and genes involved in pa thways tha t pr omote cell pr oliferation. Thus, altered expression of miRNAs dri v en by ER m utations likel y r epr esents an important mechanism by which ER mutant cells achie v e increased proliferation.
Another possib le e xplanation for the increased proliferation in ER mutant cells is that altered miRNA expression impacts the activity of important signaling pathways that pr omote pr olifera tion. miR-301b, which is down-regula ted in ER mutant cells, directly targets the PRKD3 gene, which codes for protein kinase D3 (PKD3), a regulator of cell signaling pathways that promote cell proliferation. PRKD3 is up-regulated in a mutant-specific fashion and altering the activity of miR-301b significantly altered the RNA le v els of PRKD3 . PKD3 promotes phosphorylation of se v eral components of cell signaling pathways which dri v e proliferation in breast and other cancer types (39)(40)(41)(42). We found that increased proliferation in ER mutant cells can be significantly reduced when PKD3 is inhibited either genetically by siRNA knock-down, or pharmacologically by treatment with the PKD inhibitor CRT0066101. We were surprised to find that miR-301b perturbations did not significantly impact protein le v els of PKD3. The lack of correlation between RNA and protein is commonly observed in the human proteome and could be due to se v eral possibilities ( 69 ). For example, reduction in PRKD3 RNA by miR-301b might be compensated by an increase in translation efficiency or an increase in PDK3 half life. Regardless of the mechanism of protein compensation, the lack of miR-301b effects on PKD3 protein le v els indicates that PRKD3 targeting is unlikely the driving force behind miR-301b's antiproliferati v e ability. Ev en though miR-301b may not be primarily working through PKD3, our findings show that PKD3 inhibition is a promising option for future therapeutics, with CRT0066101 having already been shown to inhibit tumor growth both in vitro and in vivo in se v eral other cancer types (62)(63)(64)(65).
miRNAs that are differentially expressed in ER mutant cells could produce varying effects that are dependent on ER mutant status. miR-301b, for example, has been shown in other settings, including triple negati v e breast cancer, to pr omote pr oliferation ( 52 , 53 ). Howe v er, we found that it has the opposite effect in ER mutant cells where increasing its activity completely abolished proliferation. These findings not only provide evidence of a significant role for miR-NAs in establishing ER mutant effects, but also indicate that some miRNAs are utilized in a unique fashion in ER mutant cells. The effect of miRNAs is highly cell contextdependent and this observation could be explained by the altered transcriptional profile seen in ER mutant cells compared to WT cells. The differential effects could result in a different set of miR-301b target genes expressed in ER mutant cells than ar e expr essed in WT cells, leading to a different overall effect of miR-301b in these cells.
We validated the mutant ER-dri v en differential e xpression of miRNAs by identifying differentially expressed miRNAs in PDX models of mutant or WT ER-positi v e breast cancer. We found that many of the same, as well as many additional, miRNAs are differentially expressed in Y537S PDX models as those identified in our T-47D cell line models. Of note, miR-301b, miR-331, and let-7f were all differ entially expr essed in the same direction in PDX Y537S models as in T-47D cells. This consistency between cell line and PDX models demonstrates that differential expression of miRNAs has the potential to play a significant role in mutant ER pathology in patients.
Overall our findings show that certain miRNAs exhibit consistent differ ential expr ession in mutant ER br east cancer models and are capable of regulating key genes and pathways that can be targeted to successfully inhibit breast cancer growth. These results also help identify additional factors and signaling pathways that could be effecti v e drug targets to treat hormone therapy-resistant ER mutant breast cancers. Additionally, the use of miRNA mimics and inhibitors to directly alter the activity of a specific miRNA has recently become an exciting new therapeutic option curr ently being explor ed. Our finding that many miRNAs ar e differ entially r egulated in the ER mutant breast cancer setting suggests that the direct targeting of these miR-NAs through mimics and inhibitors could also r epr esent a promising thera peutic a pproach in treating this hormone ther ep y-r esistant disease.

DA T A A V AILABILITY
All raw and processed sequencing data generated in this study have been submitted to the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/ ) under the following accession number: GSE214580.